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PubMed2003

Accuracy of open tray implant impressions: an in vitro comparison of stock versus custom trays.

STATEMENT OF PROBLEM: The accuracy of an implant fixture-level impression is affected by the type of impression tray used. PURPOSE: The purpose of this in vitro study was to investigate the accuracy of open tray implant impressions comparing polycarbonate stock impression trays and rigid custom-made impression trays to make implant fixture-level impressions. MATERIAL AND METHODS: Gold cylinder pairs, splinted by gold bars (reference frameworks) were constructed on an aluminum typodont. Polyether impressions were made of 2 pairs of Brånemark 3.75-mm diameter fixtures mounted in an aluminium typodont, with 3 stock impression trays, 3 close-fit custom trays, and 3 spaced custom impression trays, by use of an open tray technique. The casts produced were assessed for accuracy by attaching the reference frameworks with alternate single screws and measuring the vertical fit discrepancy of these reference frameworks to the analogs within the working cast using a traveling microscope. Comparison of gap dimensions by tray type was performed with a nonparametric Kruskal-Wallis analysis of variance (ANOVA) followed by pair-wise Mann-Whitney U tests. To adjust for multiple comparisons in the post-ANOVA contrasts (P<.02). Comparison of gap dimensions between anterior and posterior regions were performed with Mann-Whitney U tests (P <.05). RESULTS: The results showed that the mean fit accuracy, as measured by vertical fit discrepancy, of casts from the stock trays (23 +/- 20 microm) were statistically significantly less (P<.001) than the spaced custom trays (12 +/- 10 microm) or close fit custom trays (11 +/- 10 microm). The difference in median gap size for analogs with a 20-mm separation was 10 microm. CONCLUSION: Within the limits of this in vitro study, rigid custom trays produced significantly more accurate impressions than the polycarbonate stock trays. The stock trays used in this study could not produce accurate impressions consistently. For analogs with a 20-mm separation, there was a difference in medians of 10 microm in accuracy between the stock and custom trays.

Analysis of Variance↗
PubMed2003

Biaxial flexural strength, elastic moduli, and x-ray diffraction characterization of three pressable all-ceramic materials.

STATEMENT OF PROBLEM: Before the release of an advanced ceramic material, independent assessment of its strength, elastic modulus, and phase composition is necessary for comparison with peer materials. PURPOSE: This study compared the biaxial flexural strength, elastic moduli, and crystalline phases of IPS Empress and Empress 2 with a new experimental ceramic. MATERIAL AND METHODS: Twenty standardized disc specimens (14 x 1.1 mm) per material were used to measure the biaxial strength. With a universal testing machine, each specimen was supported on 3 balls and loaded with a piston at a crosshead speed of 0.5 mm/min until fracture. Three standardized bars (30 x 12.75 x 1.1 mm) for each material were prepared and excited with an impulse tool. The resonant frequencies (Hz) of the bars were used to calculate the elastic moduli with the equation suggested by the standard ASTM (C 1259-94). X-ray diffraction with Cu Kalpha at a diffraction angle from 20 to 40 degrees was used to identify the crystalline phases by means of a diffractometer attached to computer software. The data were analyzed with 1-way analysis of variance followed by pairwise t tests (P<.05). RESULTS: Mean biaxial strengths were 175 +/- 32, 407 +/- 45, and 440 +/- 55 MPa for IPS Empress, Empress 2, and the experimental ceramic, respectively. Elastic modulus results were 65, 103, and 91 GPa for the same materials, respectively. There was no significant difference in strength and elastic modulus between Empress 2 and the experimental ceramic. Both materials demonstrated a significantly higher elastic modulus and strength than IPS Empress. X-ray diffraction revealed leucite as the main crystalline phase for IPS Empress and lithium disilicate for both Empress 2 and the experimental ceramic. CONCLUSION: Within the limitations of this study, the improved mechanical properties of Empress 2 and experimental ceramic over those of IPS Empress were attributed to the nature and amount of their crystalline content lithium disilicate.

Aluminum Silicates↗
PubMed2003

Split-frame implant prosthesis designed to compensate for mandibular flexure: a clinical report.

When an edentulous mandible is restored with 4 or more implants connected by a metal bar and retained with screws, mandibular flexure may cause screw loosening and unnecessary stresses and strains on the prosthesis and implants. Separating the prosthesis at the midline can relieve these stresses and strains. This article describes the separation of a hybrid mandibular denture at the midline.

Dental Alloys↗
PubMedDate not supplied

Open surgical release for frozen shoulder: surgical findings and results of the release.

Over a 5-year period, 75 shoulders that met Codman's criteria for primary frozen shoulder were treated. Nine patients improved with nonoperative treatment, and the remaining 66 patients underwent manipulation under anesthesia. The shoulders in 41 patients successfully released with manipulation. Those in 25 failed to release with manipulation, and therefore, these patients underwent open surgical release of the contracted shoulder. We reviewed the cases of all of the surgically treated patients at 19.52 months' average follow-up, using the history and clinical examination technique recommended by the American Shoulder and Elbow Surgeons. The surgical findings in this group of 25 patients showed a consistent alteration in the rotator interval and coracohumeral ligament. The rotator interval was obliterated, and the coracohumeral ligament was transformed into a tough contracted band. The histology of this contracture was examined in 12 patients and consisted of a dense matrix of type III collagen populated with fibroblasts and myofibroblasts. The contracted coracohumeral ligament was excised with immediate release of the external rotation deficit. Pain scores on visual analogue scale improved from 8.28 to 2.0. The average score for function, with a maximum score of 30, improved from 6.08 to 18.9. Twenty patients had excellent or good results, and 3 had fair results. The shoulders of 2 patients failed to improve: 1 was an insulin-dependent patient with diabetes, and 1 had severe bilateral Dupuytren's contractures. The results in the patients without diabetes were very satisfactory, with visual analogue scale scores of pain decreasing from 8.4 to 1.1, function increasing from 6.4 to 20.1, flexion increasing from an average of 96 degrees to an average of 131 degrees, and external rotation increasing from an average of 10.0 degrees to an average of 46.7 degrees. Surgical release of frozen shoulder is a useful option in those few patients with severe disease whose shoulders fail to release with manipulation under anesthesia. Caution should be used in insulin-dependent patients with diabetes.

Adult↗
PubMed2005

Cyclization of short DNA fragments and bending fluctuations of the double helix.

Cloutier and Widom [Cloutier, T. E. & Widom, J. (2004) Mol. Cell 14, 355-362] recently reported that the cyclization efficiency of short DNA fragments, about 100 bp in length, exceeds theoretical expectations by three orders of magnitude. In an effort to resolve this discrepancy, we tried modifying the theory. We investigated how the distribution of the angles between adjacent base pairs of the double helix affects the cyclization efficiency. We found that only the incorporation of sharp kinks in the angle distribution provides the desired increase of the cyclization efficiency. We did not find a model, however, that fits all cyclization data for DNA fragments of different lengths. Therefore, we carefully reinvestigated the cyclization of 100-bp DNA fragments experimentally and found their cyclization efficiency to be in remarkable agreement with the traditional model of DNA bending. We also found an explanation for the discrepancy between our results and those of Cloutier and Widom.

Base Pairing↗
PubMed2006

Flexibly varying folding mechanism of a nearly symmetrical protein: B domain of protein A.

The folding pathway of the B domain of protein A is the pathway most intensively studied by computer simulations. Recent systematic measurement of Phi values by Sato et al., however, has shown that none of the published computational predictions is consistent with the detailed features of the experimentally observed folding mechanism. In this article we use a statistical mechanical model of folding to show that sensitive dependence of multiple transition state ensembles on temperature and the denaturant concentration is the key to resolving the inconsistency among simulations and the experiment. Such sensitivity in multiple transition state ensembles is a natural consequence of symmetry-breaking in a nearly symmetrical protein.

Dimerization↗
PubMed2006

Flexible phenylalanine-glycine nucleoporins as entropic barriers to nucleocytoplasmic transport.

Natively unfolded phenylalanine-glycine (FG)-repeat domains are alleged to form the physical constituents of the selective barrier-gate in nuclear pore complexes during nucleocytoplasmic transport. Presently, the biophysical mechanism behind the selective gate remains speculative because of a lack of information regarding the nanomechanical properties of the FG domains. In this work, we have applied the atomic force microscope to measure the mechanical response of individual and clusters of FG molecules. Single-molecule force spectroscopy reveals that FG molecules are unfolded and highly flexible. To provide insight into the selective gating mechanism, an experimental platform has been constructed to study the collective behavior of surface-tethered FG molecules at the nanoscale. Measurements indicate that the collective behavior of such FG molecules gives rise to an exponentially decaying long-range steric repulsive force. This finding indicates that the molecules are thermally mobile in an extended polymer brush-like conformation. This assertion is confirmed by observing that the brush-like conformation undergoes a reversible collapse transition in less polar solvent conditions. These findings reveal how FG-repeat domains may simultaneously function as an entropic barrier and a selective trap in the near-field interaction zone of nuclear pore complexes; i.e., selective gate.

Active Transport, Cell Nucleus↗
PubMed2001

Flexibility of the Kir6.2 inward rectifier K(+) channel pore.

Interactions of sulfhydryl reagents with introduced cysteines in the pore-forming (Kir6.2) subunits of the K(ATP) channel were examined. 2-Aminoethyl methanethiosulfonate (MTSEA(+)) failed to modify Cd(2+)-insensitive control-Kir6.2 channels, but rapidly and irreversibly modified Kir6.2[L164C] (L164C) channels. Although a single Cd(2+) ion is coordinated by L164C, four MTSEA(+) "hits" can occur, each sequentially reducing the single-channel current. A dimeric fusion of control-Kir6.2 and L164C subunits generates Cd(2+)-insensitive channels, confirming that at least three cysteines are required for coordination, but MTSEA(+) modification of the dimer occurs in two hits. L164C channels were not modified by bromotrimethyl ammoniumbimane (qBBr(+)), even though qBBr(+) caused voltage-dependent block (as opposed to modification) that was comparable to that of MTSEA(+) or 3-(triethylammonium)propyl methanethiosulfonate (MTSPTrEA(+)), implying that qBBr(+) can also enter the inner cavity but does not modify L164C residues. The Kir channel pore structure was modeled by homology with the KcsA crystal structure. A stable conformation optimally places the four L164C side chains for coordination of a single Cd(2+) ion. Modification of these cysteines by up to four MTSEA(+) (or three MTSPTrEA(+), or two qBBr(+)) does not require widening of the cavity to accommodate the derivatives within it. However, like the KcsA crystal structure, the energy-minimized model shows a narrowing at the inner entrance, and in the Kir6.2 model this narrowing excludes all ions. To allow entry of ions as large as MTSPTrEA(+) or qBBr(+), the entrance must widen to >8 A, but this widening is readily accomplished by minimal M2 helix motion and side-chain rearrangement.

Amino Acid Sequence↗
PubMed2002

Mutation of the myosin converter domain alters cross-bridge elasticity.

Elastic distortion of a structural element of the actomyosin complex is fundamental to the ability of myosin to generate motile forces. An elastic element allows strain to develop within the actomyosin complex (cross-bridge) before movement. Relief of this strain then drives filament sliding, or more generally, movement of a cargo. Even with the known crystal structure of the myosin head, however, the structural element of the actomyosin complex in which elastic distortion occurs remained unclear. To assign functional relevance to various structural elements of the myosin head, e.g., to identify the elastic element within the cross-bridge, we studied mechanical properties of muscle fibers from patients with familial hypertrophic cardiomyopathy with point mutations in the head domain of the beta-myosin heavy chain. We found that the Arg-719 --> Trp (Arg719Trp) mutation, which is located in the converter domain of the myosin head fragment, causes an increase in force generation and fiber stiffness under isometric conditions by 48-59%. Under rigor and relaxing conditions, fiber stiffness was 45-47% higher than in control fibers. Yet, kinetics of active cross-bridge cycling were unchanged. These findings, especially the increase in fiber stiffness under rigor conditions, indicate that cross-bridges with the Arg719Trp mutation are more resistant to elastic distortion. The data presented here strongly suggest that the converter domain that forms the junction between the catalytic and the light-chain-binding domain of the myosin head is not only essential for elastic distortion of the cross-bridge, but that the main elastic distortion may even occur within the converter domain itself.

Cardiomyopathy, Hypertrophic, Familial↗
PubMed2002

Protein unfolding: rigidity lost.

We relate the unfolding of a protein to its loss of structural stability or rigidity. Rigidity and flexibility are well defined concepts in mathematics and physics, with a body of theorems and algorithms that have been applied successfully to materials, allowing the constraints in a network to be related to its deformability. Here we simulate the weakening or dilution of the noncovalent bonds during protein unfolding, and identify the emergence of flexible regions as unfolding proceeds. The transition state is determined from the inflection point in the change in the number of independent bond-rotational degrees of freedom (floppy modes) of the protein as its mean atomic coordination decreases. The first derivative of the fraction of floppy modes as a function of mean coordination is similar to the fraction-folded curve for a protein as a function of denaturant concentration or temperature. The second derivative, a specific heat-like quantity, shows a peak around a mean coordination of = 2.41 for the 26 diverse proteins we have studied. As the protein denatures, it loses rigidity at the transition state, proceeds to a state where just the initial folding core remains stable, then becomes entirely denatured or flexible. This universal behavior for proteins of diverse architecture, including monomers and oligomers, is analogous to the rigid to floppy phase transition in network glasses. This approach provides a unifying view of the phase transitions of proteins and glasses, and identifies the mean coordination as the relevant structural variable, or reaction coordinate, along the unfolding pathway.

Databases, Protein↗
PubMed1997

Effects of extracellular Ca2+ concentration on hair-bundle stiffness and gating-spring integrity in hair cells.

When a hair cell is stimulated by positive deflection of its hair bundle, increased tension in gating springs opens transduction channels, permitting cations to enter stereocilia and depolarize the cell. Ca2+ is thought to be required in mechanoelectrical transduction, for exposure of hair bundles to Ca2+ chelators eliminates responsiveness by disrupting tip links, filamentous interstereociliary connections that probably are the gating springs. Ca2+ also participates in adaptation to stimuli by controlling the activity of a molecular motor that sets gating-spring tension. Using a flexible glass fiber to measure hair-bundle stiffness, we investigated the effect of Ca2+ concentration on stiffness before and after the disruption of gating springs. The stiffness of intact hair bundles depended nonmonotonically on the extracellular Ca2+ concentration; the maximal stiffness of approximately 1200 microN.m-1 occurred when bundles were bathed in solutions containing 250 microM Ca2+, approximately the concentration found in frog endolymph. For cells exposed to solutions with sufficient chelator capacity to reduce the Ca2+ concentration below approximately 100 nM, hair-bundle stiffness fell to approximately 200 microN.m-1 and no longer exhibited Ca2+-dependent changes. Because cells so treated lost mechanoelectrical transduction, we attribute the reduction in bundle stiffness to tip-link disruption. The results indicate that gating springs are not linearly elastic; instead, they stiffen with increased strain, which rises with adaptation-motor activity at the physiological extracellular Ca2+ concentration.

Acoustic Maculae↗
PubMed1998

Flexibility of helix 2 in the human glutathione transferase P1-1. time-resolved fluorescence spectroscopy.

Time-resolved fluorescence spectroscopy and site-directed mutagenesis have been used to probe the flexibility of alpha-helix 2 (residues 35-46) in the apo structure of the human glutathione transferase P1-1 (EC 2.5.1.18) as well as in the binary complex with the natural substrate glutathione. Trp-38, which resides on helix 2, has been exploited as an intrinsic fluorescent probe of the dynamics of this region. A Trp-28 mutant enzyme was studied in which the second tryptophan of glutathione transferase P1-1 is replaced by histidine. Time-resolved fluorescence data indicate that, in the absence of glutathione, the apoenzyme exists in at least two different families of conformational states. The first one (38% of the total population) corresponds to a number of slightly different conformations of helix 2, in which Trp-38 resides in a polar environment showing an average emission wavelength of 350 nm. The second one (62% of the total population) displays an emission centered at 320 nm, thus suggesting a quite apolar environment near Trp-38. The interconversion between these two conformations is much slower than 1 ns. In the presence of saturating glutathione concentrations, the equilibrium is shifted toward the apolar component, which is now 83% of the total population. The polar conformers, on the other hand, do not change their average decay lifetime, but the distribution becomes wider, indicating a slightly increased rigidity. These data suggest a central role of conformational transitions in the binding mechanism, and are consistent with NMR data (Nicotra, M., Paci, M., Sette, M., Oakley, A. J., Parker, M. W., Lo Bello, M., Caccuri, A. M., Federici, G., and Ricci, G. (1998) Biochemistry 37, 3020-3027) and pre-steady state kinetic experiments (Caccuri, A. M., Lo Bello, M., Nuccetelli, M., Nicotra, M., Rossi, P., Antonini, G., Federici, G., and Ricci, G. (1998) Biochemistry 37, 3028-3034) indicating the existence of a pre-complex in which GSH is not firmly bound to the active site.

Binding Sites↗
PubMed1998

Flexibility of the neck region of the rieske iron-sulfur protein is functionally important in the cytochrome bc1 complex.

The crystal structure of the mitochondrial cytochrome bc1 complex suggests that movement of the extramembrane (head) domain of the Rieske iron-sulfur protein (ISP) is involved in electron transfer. Such movement requires flexibility in the neck region of ISP. To test this hypothesis, Rhodobacter sphaeroides mutants expressing His-tagged cytochrome bc1 complexes with altered ISP necks (residues 39-48) were generated and characterized. Mutants with increased rigidity of the neck, generated by a double-proline substitution at Ala-46 and Ala-48 (ALA-PLP) or by a triple-proline substitution of ADV at residues 42-44 (ADV-PPP), have retarded (50%) or no photosynthetic growth, respectively. However, the mutant with a shortened neck, generated by deleting ADV (DeltaADV), has a photosynthetic growth rate comparable to that of complement cells, indicating that the length of the ISP neck is less critical than its flexibility in support of photosynthetic growth. The DeltaADV and ALA-PLP mutant membranes have 10 and 30% of the cytochrome bc1 complex activity found in the complement membrane, respectively, whereas the ADV-PPP mutant membrane contains no cytochrome bc1 complex activity. The loss of cytochrome bc1 complex activity in the DeltaADV membrane is attributed to improper docking of the head domain of ISP on cytochrome b, as indicated by a drastic change in the EPR characteristics of the Rieske iron-sulfur cluster. The loss of cytochrome bc1 complex activity in the ALA-PLP and ADV-PPP mutant membranes results from the decreased mobility of the ISP head domain due to the increased rigidity of the ISP neck. The ALA-PLP mutant complex has a larger activation energy than the wild-type complex, suggesting that movement of the head domain decreases the activation energy barrier of the cytochrome bc1 complex. Using the conditions developed for the isolation of the His-tagged complement cytochrome bc1 complex, a two-subunit complex (cytochromes b and c1) was obtained from the DeltaADV and ADV-PPP mutants, indicating that mutations at the neck region of ISP weaken the interactions among cytochrome b, ISP, and subunit IV.

Amino Acid Sequence↗
PubMed2000

In vitro studies on the maintenance of transcription-induced stress by histones and polyamines.

Several factors were evaluated to determine their role in facilitating the presence of transcription-induced stresses in a circular DNA. Transcription was done with T7 RNA polymerase in the presence of E. coli topoisomerase I and closed circular DNA. Positive stress was observed in hypotonic conditions or when one of the polyamines, spermidine or spermine, were present. Polycations such as polylysine, polyarginine, histone H1, histones H2A-H2B, and protamine were observed to induce minimal positive stress. It is known that polyamines influence DNA structure by causing both self-association and sequence-specific structural alterations (polyamine-induced localized bending). Experimental evidence indicates that the likely cause of the positive stress is the induced bending. In order to evaluate protein-mediated bending, transcription was done on nucleosomes. A minimum of three nucleosomes on a DNA of 6055 bp was sufficient to generate very high levels of positive stress. Histones H3-H4 in the absence of H2A-H2B were responsible for this effect. Since these histones by themselves are able to maintain negative coils on DNA, it is concluded that protein-mediated bending is yet another mechanism for placing rotational restriction on DNA. The bending of DNA by either polyamines or histones is an effective mechanism for promoting transcription-induced stresses at physiological ionic strength.

Animals↗
PubMed2000

Actin activates a cryptic dimerization potential of the vinculin tail domain.

The tail domain of vinculin (V(t)) is an actin binding module containing two regions that interact with F-actin. Although intact V(t) purified from a bacterial expression system is a globular monomer, each actin binding region dimerizes when expressed individually, suggesting the presence of cryptic self-association sites whose exposure is regulated. We show that actin modulates V(t) self-association by inducing or stabilizing a conformational change in V(t) that allows dimerization. Chemical cross-linking studies implicate one of the actin binding regions in mediating dimerization in the presence of actin. Actin-induced V(t) dimers may play a role in the filament cross-linking activity of this protein. The V(t) dimers induced by actin are biochemically distinct from the V(t) dimers and higher oligomers induced by acidic phospholipids such as phosphatidylinositol 4,5-bisphosphate, suggesting structural differences in V(t) bound to these two ligands that may provide a mechanistic basis for inhibition of F-actin binding by phosphatidylinositol 4,5-bisphosphate. The ability of actin to regulate the dimerization state of an actin binding protein suggests that, rather than serving a passive structural role, actin filaments may directly participate in signal transduction and other cellular events that are known to depend on cytoskeletal integrity.

Actins↗
PubMed2000

Structure of Cdc4p, a contractile ring protein essential for cytokinesis in Schizosaccharomyces pombe.

The Schizosaccharomyces pombe Cdc4 protein is required for the formation and function of the contractile ring, presumably acting as a myosin light chain. By using NMR spectroscopy, we demonstrate that purified Cdc4p is a monomeric protein with two structurally independent domains, each exhibiting a fold reminiscent of the EF-hand class of calcium-binding proteins. Although Cdc4p has one potentially functional calcium-binding site, it does not bind calcium in vitro. Three variants of Cdc4p containing single point mutations responsible for temperature-sensitive arrest of the cell cycle at cytokinesis (Gly-19 to Glu, Gly-82 to Asp, and Gly-107 to Ser) were also characterized by NMR and circular dichroism spectroscopy. In each case, the amino acid substitution only leads to small perturbations in the conformation of the protein. Furthermore, thermal unfolding studies indicate that, like wild-type Cdc4p, the three mutant forms are all extremely stable, remaining completely folded at temperatures significantly above those causing failure of cytokinesis in intact cells. Therefore, the altered phenotype must arise directly from a disruption of the function of Cdc4p rather than indirectly through a disruption of its overall structure. Several mutant alleles of Cdc4p also show interallelic complementation in diploid cells. This phenomenon can be explained if Cdcp4 has more than one essential function or, alternatively, if two mutant proteins assemble to form a functional complex. Based on the structure of Cdc4p, possible models for interallelic complementation including interactions with partner proteins and the formation of a myosin complex with Cdc4p fulfilling the role of both an essential and regulatory light chain are proposed.

Cell Cycle Proteins↗
PubMed2003

Interacting helical surfaces of the transmembrane segments of subunits a and c' of the yeast V-ATPase defined by disulfide-mediated cross-linking.

Proton translocation by the vacuolar (H+)-ATPase (or V-ATPase) has been shown by mutagenesis to be dependent upon charged residues present within transmembrane segments of subunit a as well as the three proteolipid subunits (c, c', and c"). Interaction between R735 in TM7 of subunit a and the glutamic acid residue in the middle of TM4 of subunits c and c' or TM2 of subunit c" has been proposed to be essential for proton release to the luminal compartment. In order to determine whether the helical face of TM7 of subunit a containing R735 is capable of interacting with the helical face of TM4 of subunit c' containing the essential glutamic acid residue (Glu-145), cysteine-mediated cross-linking between these subunits in yeast has been performed. Cys-less forms of subunits a and c' as well as forms containing unique cysteine residues were constructed, introduced together into a strain disrupted in both endogenous subunits, and tested for growth at neutral pH, for assembly competence and for cross-linking in the presence of cupric-phenanthroline by SDS-PAGE and Western blot analysis. Four different cysteine mutants of subunit a were each tested pairwise with ten different unique cysteine mutants of subunit c'. Strong cross-linking was observed for the pairs aS728C/c'I142C, aA731C/c'E145C, aA738C/c'F143C, aA738C/c'L147C, and aL739C/c'L147C. Partial cross-linking was observed for an additional 13 of 40 pairs analyzed. When arrayed on a helical wheel diagram, the results suggest that the helical face of TM7 of subunit a containing Arg-735 interacts with the helical face of TM4 of subunit c' centered on Val-146 and bounded by Glu-145 and Leu-147. The results are consistent with a possible rotational flexibility of one or both of these transmembrane segments as well as some flexibility of movement perpendicular to the membrane.

Amino Acid Substitution↗
PubMed2004

Structural and dynamics studies of the D54A mutant of human T cell leukemia virus-1 capsid protein.

The human T cell leukemia virus and the human immunodeficiency virus share a highly conserved, predominantly helical two-domain mature capsid (CA) protein structure with an N-terminal beta-hairpin. Despite overall structural similarity, differences exist in the backbone dynamic properties of the CA N-terminal domain. Since studies with other retroviruses suggest that the beta-hairpin is critical for formation of a CA-CA interface, we investigated the functional role of the human T cell leukemia virus beta-hairpin by disrupting the salt bridge between Pro(1) and Asp(54) that stabilizes the beta-hairpin. NMR (15)N relaxation data were used to characterize the backbone dynamics of the D54A mutant in the context of the N-terminal domains, compared with the wild-type counterpart. Moreover, the effect of the mutation on proteolytic processing and release of virus-like particles (VLPs) from human cells in culture was determined. Conformational and dynamic changes resulting from the mutation were detected by NMR spectroscopy. The mutation also altered the conformation of mature CA in cells and VLPs, as reflected by differential antibody recognition of the wild-type and mutated CA proteins. In contrast, the mutation did not detectably affect antibody recognition of the CA protein precursor or release of VLPs assembled by the precursor, consistent with the fact that the hairpin cannot form in the precursor molecule. The particle morphology and size were not detectably affected. The results indicate that the beta-hairpin contributes to the overall structure of the mature CA protein and suggest that differences in the backbone dynamics of the beta-hairpin contribute to mature CA structure, possibly introducing flexibility into interface formation during proteolytic maturation.

Capsid Proteins↗